The following abbreviations will be used in this disclosure:
______________________________________ GAL Galactose GALT Galactose-1-Phosphate Uridyl Transferase GAL-1-P Galactose-1-Phosphate UDPG Uridine Diphosphoglucose UDP-GAL Uridine Diphosphogalactose G-1-P Glucose-1-Phosphate PGM Phosphoglucomutase G-6-P Glucose-6-Phosphate NADP Nicotinamide Adenine Dinucleotide Phosphate NADPH Nicotinamide Adenine Dinucleotide, Reduced G-6-PDH Glucose-6-Phosphate Dehydrogenase 6-PGA 6-Phosphogluconate 6-PGD 6-Phosphogluconate Dehydrogenase R-5-P Ribulose-5-Phosphate TRIS Tris(hydroxyethyl)aminomethane EDTA Ethylenediaminetetraacetic Acid EtOH Ethanol MeOH Methanol DTT Dithiothreitol ______________________________________
The incidence of galactosemia is about 1 in 80,000 births in the U.S. If the disease is detected in the first few days of life, a newborn can be placed on special galactose-free diets to prevent the severe symptoms of the disease. Galactose is generally derived from lactose, which is the main carbohydrate in milk. When galactosemia is not detected in the first few days of life, it may cause liver damage, cataracts, mental retardation and, occasionally, death.
Accordingly, it is very desirable to test reliably for galactosemia in newborns, enabling the appropriate treatment measures to be timely taken.
There are two commonly used methods to detect galactosemia in newborns. One way is to measure the metabolites (GAL and GAL-1-P), which accumulate in the neonate's blood. This accumulation can occur only after dietary exposure of the infant to milk. Levels of the metabolites increase and remain high due to enzyme deficiency. An alternative method measures galactose-1-phosphate uridyl transferase (E.C. 2.7.7.12; GALT) activity, the most common enzyme deficiency in galactosemia. The following Table 1 compares and contrasts these two methods:
TABLE 1 __________________________________________________________________________ PROPERTY TOTAL GALACTOSE ASSAY GALT ASSAY __________________________________________________________________________ ANALYTE GAL & GAL-1-P GALT ACTIVITY DETECTS ALL FORMS OF ONLY GALT DEFICIENCY GALACTOSEMIA PATIENT MUST BE EXPOSED TO GAL IN ASSAY REQUIRES THREE REQUIREMENTS DIET ENZYMES FROM SAMPLE SAMPLE GOOD ENZYME(S) DEGRADE IN STABILITY HEAT DURING TRANSPORT UNITS mg/dl GAL .mu.mol hour.sup.-1 mL.sup.-1 or % activity of a high control __________________________________________________________________________
There are three enzymes whose deficiency leads to galactosemia: GALT, galactokinase, and UDPG galactose-4-epimerase. Classic galactosemia (GALT deficiency) is the most common. GALT deficiency is an inborn error of metabolism transmitted through an autosomal recessive gene. Untreated galactokinase-deficient patients suffer from cataracts, but other debilitating symptoms do not occur. The epimermase deficiency is quite rare and, in most cases, there are few clinical symptoms associated with epimerase deficiency. For a review of screening and diagnosis of galactosemia, see Beutler (1991).
In 1966, Beutler and Baluda reported a spot screening test for GALT deficiency. In that procedure, NADP is reduced to NADPH as a result of a series of enzymatic reactions. The assay is very simple. Sample is mixed with reagent and allowed to incubate for a certain period of time. Drops of the reaction mixture are removed and spotted onto filter paper. NADPH fluorescence is then detected using a black light (long-wave UV light). If GALT activity is not present in a blood sample, NADP is not reduced to NADPH and no fluorescence results. In this assay, GALT catalyses the reaction: ##STR1## The above reaction is coupled to the following enzyme system to yield a fluorescent NADPH product: ##STR2## In the Beutler and Baluda procedure, a reaction mixture containing GAL-1-P, UDPG, and NADP is mixed with a blood sample and incubated at 37.degree. C. Since GALT is located in the red blood cell, whole blood or a dried blood spot is generally the sample of choice. Drops from the reaction mixture are spotted onto filter paper at time zero and at various intervals during the incubation. The spots are then visualized under UV light. Fluorescence indicates the presence of GALT activity. The fluorescence will range from bright for normal blood to no fluorescence for GALT deficient samples. If the blood is from a heterozygote for GALT deficiency, a subdued fluorescence results. Three enzymes required for the reaction are provided by the patient's blood: PGM, G-6-PDH and 6-PGD. This paper-spot assay has been commercialized by Sigma Diagnostics, St. Louis, Mo. (Procedure No. 195).
This assay has two major flaws. The first is that this fluorescent spot test may miss samples that are galactose kinase deficient. The second flaw is that GALT is unstable to heating, and samples stored at high temperatures may give false positive results. It has recently been proposed (Berry 1987) that this problem can be largely overcome by the addition of DTT to the reaction mixture. The Beutler and Baluda assay has also been adapted to an autoanalyzer (Hochella and Hill 1969) for screening samples collected as dried blood spots on filter paper. A reagent similar to the Beutler and Baluda reagent is added to eluted blood samples. The samples are loaded on an autoanalyzer and measured twice (at time zero and an hour later). The difference in fluorescence intensity of the two readings is used as evidence of GALT activity. The authors emphasized that their assay could not give results expressed in international enzyme units, but was rather designed for a screening method to give a yes/no answer.
Methods which measure the NADPH product optically have also been reported. A kinetic micro-spectrophotometric assay of whole blood was done in an LKB Reaction Rate Analyzer (Pesce et al. 1977). In this assay a modified Beutler reagent is mixed with whole blood and the rate of NADPH formation is measured spectrophotometrically. Actual enzyme units can be calculated after hemoglobin concentration measurement.
The GALT assay has also been used to confirm the results of total (GAL plus GAL-1-P) galactose assays (Greenberg et al. 1989).
The use of microtiter plates and fluorescent readout has been recently described (Yamaguchi et al. 1989) in which total galactose (GAL plus GAL-1-P) was measured in blood dried on filter paper. A punched-out spot was first extracted with a methanol:acetone:water mixture, during which hemoglobin and other proteins were denatured. Water was then added and the extract transferred to an assay plate where a galactose dehydrogenase enzyme reagent converted galactose and NAD to products. The NADH product concentration, which is proportional to the original galactose concentration in the sample, was measured fluorometrically.
A manual fluorometric assay for GALT activity has also been reported (Frazier, Clemons, and Kirkman 1992). A dried blood spot on filter paper was mixed with a modified Beutler reagent and incubated for various amounts of time; after which a portion of the sample-reagent mixture was transferred to a holding reagent, which stopped the reaction. A Turner fluorometer was used to measure fluorescence.
Biotinidase deficiency, also termed late-onset or juvenile multiple-carboxylase deficiency, is a rare genetic disease inherited as an autosomal recessive trait (Pettit and Wolf, 1991; Pitkanen and Tuuminen, 1992). Individuals having biotinidase deficiency exhibit a variety of symptoms, such as seizures, hypotonia, alopecia, skin rash, hearing loss, developmental delay, keto-lactic acidosis, and organic aciduria (Wolf et al., 1985), making it difficult to diagnose the disease clinically. Biotinidase deficiency can be of three types: (i) complete or profound biotinidase deficiency (less than 10% of mean normal adult activity), (ii) partial biotinidase deficiency (10-30% of mean adult activity), and (iii) transient biotinidase deficiency (little or no activity in the original newborn blood specimen and normal activity in a requested repeat filter-paper specimen). Pharmacological doses of oral biotin (10 mg/day) may alleviate symptoms and, if initiated early, may prevent them (Wolf et al., 1983; Wolf and Heard, 1989).
The incidence of biotinidase deficiency varies widely. The frequency of 1:33,000 for profound biotinidase deficiency in Massachusetts (Lawler et al., 1992) is similar to the incidence of 1:54,000 in Quebec (Dunkel et al., 1989). The mean frequency for profound biotinidase deficiency from worldwide screening experience is 1:137,000; but there is a wide range among these screening programs, varying from 1:33,000 in New Zealand to &lt;1:500,000 in Illinois (Wolf and Heard, 1990).
Biotinidase deficiency can be identified in newborn infants by a simple and inexpensive screening test which was first demonstrated by Wolf and his group (Wolf et al., 1985) and repeated by the screening program in Quebec (Dunkel et. al., 1989) and a number of others (Wolf and Heard, 1990). The screening test is easily accommodated in newborn screening programs.
Biotinidase activity can be determined using a modification of the colorimetric assay described by Knappe et al. (1963) which measures the release of p-aminobenzoate (p-ABA) from the artificial substrate, biotinyl-p-aminobenzoic acid (B-pABA), an analogue of biocytin. Further modification of this assay has enabled quantitative and qualitative determination of biotinidase activity in whole-blood filter-paper spots (Heard et al., 1984; Dove Pettit et al., 1989). The main drawback of the colorimetric assay is that it requires visual inspection, which may be subjective. The quantitative determination of biotinidase activity requires a serum or plasma as the sample, often not available where only blood spots are available or practical.
Another method is to separate p-ABA from B-pABA by high performance liquid chromatography in which p-ABA concentration is determined fluorometrically (Hayakawa and Oizumi, 1986). An advantage of this method is that p-ABA can be distinguished from sulfonamides and other interferents. However, this method is not practical for clinical screening purposes.
Biocytin, the natural substrate of biotinidase, is used in several methods. One method detects the activation and increase of propionyl-CoA carboxylase activity in holocarboxylase synthetase-deficient fibroblasts by measuring the biotin that is liberated from biocytin by biotinidase (Weiner et al., 1985). Other methods observe the growth of biotin-dependent bacteria or protozoa from the biotin that is liberated from biocytin (Thoma and Peterson, 1954; Wright et al., 1954; Baker et al., 1989). An additional method is a radioassay in which liberated [.sup.14 C]biotin is separated from [.sup.14 C]biocytin by anion-exchange chromatography (Thuy et al., 1985). These methods are laborious and require reagents which are not readily available and may need to be synthesized, making these assays impractical for use in clinical or diagnostic settings.
One microplate-based fluorometric assay measures the release of 6-aminoquinoline from the artificial substrate biotinyl-6-aminoquinoline (Wastell et al., 1984; Pitkanen and Tuuminen, 1992). The disadvantage of this method is that it utilizes serum or plasma. Another fluorometric assay uses biocytin and measures the release of lysine, which complexes with 1,2-diacetylbenzene to give a fluorescent product. The drawbacks of this method are that it uses serum that must be dialyzed extensively, and that it is not microplate-based.
The microplate-based fluorometric assay seems the most promising for screening due to its low cost and robustness. However, the main drawback is that it requires serum as a sample.
With the foregoing background in mind, it is desirable to be able to perform an enzyme assay (such as a GALT or biotinidase assay) in a single analysis vessel, and to have an assay protocol which can be used with conveniently handled samples. It is also advantageous to be able to analyze a sample without having to transfer it from one vessel to another prior to analysis.
It is also desirable to be able to perform a quantitative enzyme assay (such as a GALT or biotinidase assay) using a single reading by the operator.
Problems associated with enzyme assays are errors where increases in enzyme activity occur due to more or less sample. Accordingly, it is beneficial to produce an assay protocol capable of balancing such effects.
Because enzyme deficient patients are so rare, it is difficult to obtain human blood for making control materials. Accordingly, it is desirable to be able to use commonly available substances which can be used to make appropriately precise enzyme-deficient controls.
Although described in the context of an enzyme assay for GALT or biotinidase, the present invention may be applied to solve the problems associated with other enzyme assays conducted in the presence of hemoglobin. These include other enzymes associated with the cellular and/or serum portions of a blood sample.
In view of the present disclosure and/or through the practice of the invention itself, other efficiencies, benefits and advantages, and/or the solution to other problems may become apparent to one of ordinary skill in the art.